Metering Technique for Lateral Flow Assay Devices

ABSTRACT

A diagnostic method and associated test kit for detecting an analyte residing in a test sample is provided. A sample membrane is utilized having a collection region and a detection region, the collection region having a known saturation volume for the intended test sample. A barrier is defined between the collection region and the detection region. The collection region is saturated with the test sample having a volume of less than about 100 microliters so that a known volume of the test sample is contained in the collection region. The barrier is removed from between the collection region and detection region of the membrane and a diluent is supplied to the collection region of the membrane to facilitate flow of the test sample from the collection region to the detection region of the membrane.

RELATED APPLICATION

The present application is a Divisional Application of U.S. applicationSer. No. 11/301,631 filed on Dec. 13, 2005.

BACKGROUND OF THE INVENTION

Test strips are often used for qualitative and quantitative analysis ofblood components. The test strips are sometimes constructed so that thesample application area and the detection area are stacked above oneanother in a vertical axis. However, this type of construction isassociated with a number of problems. For example, when the test stripis inserted into an instrument for measurement, the potentiallyinfectious sample material may contact parts of the optical reader andresult in contamination. Thus, spatial separation between the sampleapplication area and detection zone is often desired, i.e., lateral flowstrips. Most conventional lateral flow strips are designed for testsamples that are readily available in large quantities (e.g., urine).However, when the test sample is blood, the collection of a large samplemay cause undue pain to the patient. Thus, one technique that has beenutilized to accommodate smaller test sample volumes is to “spot” thesample directly only the membrane surface. Thereafter, a diluent is usedto wash away the test sample and carry it to the detection zone.Unfortunately, variations associated with sample transfer and diffusionof the sample to the membrane result in a flow that is largelyuncontrolled and uneven before reaching the detection zone. This mayhave an adverse affect on the accuracy of the device because the amountof analyte and/or label captured across the detection zone is notconsistent at the time of measurement.

As such, a need currently exists for a simple and efficient techniquefor metering a low volume test sample to a detection zone of a lateralflow assay device.

SUMMARY OF THE INVENTION

Objects and advantages of the invention will be set forth in part in thefollowing description, or may be obvious from the description, or may belearned through practice of the invention.

In accordance with one embodiment of the present invention, a diagnosticmethod, and test kit for practice of the method, are provided fordetecting the presence of an analyte within a test sample. A testmembrane is provided having a collection region and a detection region,the collection region having a known saturation volume for the sample tobe tested. A temporary or removable barrier is defined between thecollection region and the detection region. The collection region issaturated with a test sample having a volume of less than about 100microliters so that a known volume of the test sample is contained inthe collection region. The barrier is subsequently removed from betweenthe collection region and detection region of the membrane, and adiluent is supplied to the collection region to facilitate flow of thetest sample from the collection region to the detection region.

In one embodiment, the barrier may be defined by a soluble compositionin a defined band across the width of the membrane, with the solublecomposition present throughout the membrane in an amount to preventmigration of the test sample from the collection region to the detectionregion. The soluble composition may be dissolved by the diluent and isselected so as to not to affect detection of the analyte in the testsample.

In an alternate embodiment, the barrier may be defined by a physicaldivide or separation in the membrane, such as a channel across the widthof the membrane, wherein the step of removing the barrier includesforming a bridge across the divide so that the test sample flows fromthe collection region to the detection region with the diluent.

The invention is not limited to any particular test sample, and in oneembodiment, the test sample is whole blood. Plasma, serum, or both maybe separated from the whole blood for analysis. In the embodimentwherein a bridging member is used to provide flow of the test sampleacross a channel in the membrane, the bridging member may also functionas a blood separation filter.

The collection region may be a sub-region of a larger receiving regionof the membrane. Prior to supplying the diluent, the collection regionhaving a known saturation volume is separated from the receiving region,for example by scoring or cutting the membrane along a line that definesthe collection region.

Various means may be provided to convey the test sample to thecollection region. In one embodiment, the test sample may be depositedby the user directly onto the collection region in an amount to ensuresaturation of the collection region. In an alternate embodiment, thetest sample may be conveyed by structure defined in the membrane, suchas a channel. The channel may be disposed along an edge of thecollection region for relatively rapid intake of the test sample intothe collection region. The test sample may migrate from the channel intothe collection region and, after a sufficient time, the collectionregion is saturated with the test sample. The channel may be incommunication with a collection tip for direct intake of the test sampleinto the channel. For example, the tip may be formed at an end of thechannel and configured for placement adjacent to the skin of a user forreceipt of a test sample of blood.

In one embodiment wherein a channel is formed in the membrane forconveying the test sample, the channel need not form a boundary of thecollection region. The collection region may be separated from theregion of the membrane containing the channel by scoring or cutting themembrane prior to supplying the diluent to the collection region.

Any channel structure defined in the membrane may be formed by asuitable microfabrication technique that renders at least a portion ofthe channel essentially non-conductive to the test sample. Such atechnique may include laser ablation or applying a solvent treatment tothe membrane. In this instance, a bridging member may be used to conductthe test sample from the channel to the collection region. The channelmay be treated with a hydrophobic agent to promote rapid collection ofthe test sample within the channel.

The invention also includes any manner of test kit utilizing a membraneconfigured for practicing the method described herein.

Other features and aspects of the present invention are discussed ingreater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

A full and enabling disclosure of the present invention, including thebest mode thereof, directed to one of ordinary skill in the art, is setforth more particularly in the remainder of the specification, whichmakes reference to the appended figures in which:

FIG. 1 is a perspective view of one embodiment of a lateral flow assaydevice of the present invention.

FIG. 2 is cut-away view of the lateral flow assay device shown in FIG.1, particularly illustrating a soluble composition barrier between thecollection region and detection region of the membrane.

FIG. 3 is a perspective view of an alternate embodiment of a lateralflow assay device in accordance with aspects of the invention.

FIG. 4 is a cut-away view of the lateral flow assay device shown in FIG.3.

FIG. 5 is a top perspective view of a portion of the lateral flow assaydevice of FIG. 3 particularly illustrating use of bridging memberbetween the collection region and detection region.

FIG. 6 is a perspective view of another embodiment of a lateral flowassay device of the present invention.

Repeat use of reference characters in the present specification anddrawings is intended to represent same or analogous features or elementsof the invention.

DETAILED DESCRIPTION OF REPRESENTATIVE EMBODIMENTS Definitions

As used herein, the term “analyte” generally refers to a substance to bedetected. For instance, analytes may include antigenic substances,haptens, antibodies, and combinations thereof. Analytes include, but arenot limited to, toxins, organic compounds, proteins, peptides,microorganisms, amino acids, nucleic acids, hormones, steroids,vitamins, drugs (including those administered for therapeutic purposesas well as those administered for illicit purposes), drug intermediariesor byproducts, bacteria, virus particles and metabolites of orantibodies to any of the above substances. Specific examples of someanalytes include ferritin; creatinine kinase MB (CK-MB); digoxin;phenytoin; phenobarbitol; carbamazepine; vancomycin; gentamycin;theophylline; valproic acid; quinidine; luteinizing hormone (LH);follicle stimulating hormone (FSH); estradiol, progesterone; C-reactiveprotein; lipocalins; IgE antibodies; cytokines; vitamin B2micro-globulin; glycated hemoglobin (Gly. Hb); cortisol; digitoxin;N-acetylprocainamide (NAPA); procainamide; antibodies to rubella, suchas rubella-IgG and rubella IgM; antibodies to toxoplasmosis, such astoxoplasmosis IgG (Toxo-IgG) and toxoplasmosis IgM (Toxo-IgM);testosterone; salicylates; acetaminophen; hepatitis B virus surfaceantigen (HBsAg); antibodies to hepatitis B core antigen, such asanti-hepatitis B core antigen IgG and IgM (Anti-HBC); human immunedeficiency virus 1 and 2 (HIV 1 and 2); human T-cell leukemia virus 1and 2 (HTLV); hepatitis B e antigen (HBeAg); antibodies to hepatitis B eantigen (Anti-HBe); influenza virus; thyroid stimulating hormone (TSH);thyroxine (T4); total triiodothyronine (Total T3); free triiodothyronine(Free T3); carcinoembryoic antigen (CEA); lipoproteins, cholesterol, andtriglycerides; and alpha fetoprotein (AFP). Drugs of abuse andcontrolled substances include, but are not intended to be limited to,amphetamine; methamphetamine; barbiturates, such as amobarbital,secobarbital, pentobarbital, phenobarbital, and barbital;benzodiazepines, such as librium and valium; cannabinoids, such ashashish and marijuana; cocaine; fentanyl; LSD; methaqualone; opiates,such as heroin, morphine, codeine, hydromorphone, hydrocodone,methadone, oxycodone, oxymorphone and opium; phencyclidine; andpropoxyhene. Other potential analytes may be described in U.S. Pat. No.6,436,651 to Everhart, et al. and U.S. Pat. No. 4,366,241 to Tom et al.

As used herein, the term “test sample” generally refers to a biologicalmaterial suspected of containing the analyte. The test sample may bederived from any biological source, such as a physiological fluid,including, blood, interstitial fluid, saliva, ocular lens fluid,cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, nasalfluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menses,amniotic fluid, semen, and so forth. Besides physiological fluids, otherliquid samples may be used such as water, food products, and so forth,for the performance of environmental or food production assays. Inaddition, a solid material suspected of containing the analyte may beused as the test sample. The test sample may be used directly asobtained from the biological source or following a pretreatment tomodify the character of the sample. For example, such pretreatment mayinclude preparing plasma from blood, diluting viscous fluids, and soforth. Methods of pretreatment may also involve filtration,precipitation, dilution, distillation, mixing, concentration,inactivation of interfering components, the addition of reagents,lysing, etc. Moreover, it may also be beneficial to modify a solid testsample to form a liquid medium or to release the analyte.

Detailed Description

Reference now will be made in detail to various embodiments of theinvention, one or more examples of which are set forth below. Eachexample is provided by way of explanation of the invention, notlimitation of the invention. In fact, it will be apparent to thoseskilled in the art that various modifications and variations may be madein the present invention without departing from the scope or spirit ofthe invention. For instance, features illustrated or described as partof one embodiment, may be used on another embodiment to yield a stillfurther embodiment. Thus, it is intended that the present inventioncovers such modifications and variations as come within the scope of theappended claims and their equivalents.

The present invention is directed to a diagnostic method and associatedtest kit for detecting an analyte residing in a test sample. The methodemploys a lateral flow device that contains a membrane. The membraneincludes a collection region having a known saturation volume for thesample to be tested, and a detection region. A temporary or removablebarrier is defined between the collection region and the detectionregion. In use, the collection region is saturated with a test samplehaving a volume of less than about 100 microliters so that a knownvolume of the test sample is contained in the collection region. Thebarrier is subsequently removed from between the collection region anddetection region of the membrane, and a diluent is supplied to thecollection region to facilitate flow of the test sample from thecollection region to the detection region.

The membrane with defined collection region is particularly well suitedfor delivering a controlled volume of the test sample to the detectionregion, and is particularly effective for embodiments in which the testsample has a relatively low volume, such as less than about 100microliters, in some embodiments less than about 25 microliters, and insome embodiments, less than about 10 microliters. For example, wholeblood drops obtained from patients with a lancet from low-pain areashaving reduced nerve endings as compared to a fingertip, such as theforearm, thigh, or other alternate sites, may have a volume of fromabout 0.1 to about 5 microliters. Despite their low volume, the presentinventors have discovered that the blood drops may still be accuratelyanalyzed for the presence of an analyte using lateral flow detectiontechniques.

Referring to FIG. 1, for instance, one embodiment of a diagnostic testkit that may be formed according to the present invention will now bedescribed in more detail. As shown, the diagnostic test kit includes alateral flow assay device 20, which contains a membrane 23 optionallysupported by a rigid support material 21. In general, the membrane 23may be made from any of a variety of materials through which the testsample is capable of passing. For example, the membrane 23 may be formedfrom natural, synthetic, or naturally occurring materials that aresynthetically modified, such as polysaccharides (e.g., cellulosematerials such as paper and cellulose derivatives, such as celluloseacetate and nitrocellulose); polyether sulfone; polyethylene; nylon;polyvinylidene fluoride (PVDF); polyester; polypropylene; silica;inorganic materials, such as deactivated alumina, diatomaceous earth,MgSO₄, or other inorganic finely divided material uniformly dispersed ina porous polymer matrix, with polymers such as vinyl chloride, vinylchloride-propylene copolymer, and vinyl chloride-vinyl acetatecopolymer; cloth, both naturally occurring (e.g., cotton) and synthetic(e.g., nylon or rayon); porous gels, such as silica gel, agarose,dextran, and gelatin; polymeric films, such as polyacrylamide; and soforth. Particularly desired materials for forming the membrane 23include polymeric materials, such as nitrocellulose, polyether sulfone,polyethylene, nylon, polyvinylidene fluoride, polyester, andpolypropylene. It should be understood that the term “nitrocellulose”refers to nitric acid esters of cellulose, which may be nitrocellulosealone, or a mixed ester of nitric acid and other acids, such asaliphatic carboxylic acids having from 1 to 7 carbon atoms.

The size and shape of the membrane 23 may generally vary as is readilyrecognized by those skilled in the art. For instance, a membrane stripmay have a length of from about 10 to about 100 millimeters, in someembodiments from about 20 to about 80 millimeters, and in someembodiments, from about 40 to about 60 millimeters. The width of themembrane strip may also range from about 0.5 to about 20 millimeters, insome embodiments from about 1 to about 15 millimeters, and in someembodiments, from about 2 to about 10 millimeters. Although notrequired, the thickness of the membrane strip may be small enough toallow transmission-based detection. For example, the membrane strip mayhave a thickness less than about 500 micrometers, in some embodimentsless than about 250 micrometers, and in some embodiments, less thanabout 150 micrometers.

As stated above, the support 21 carries the membrane 23. For example,the support 21 may be positioned directly adjacent to the membrane 23 asshown in FIG. 1, or one or more intervening layers may be positionedbetween the membrane 23 and the support 21. Regardless, the support 21may generally be formed from any material able to carry the membrane 23.The support 21 may be formed from a material that is transmissive tolight, such as transparent or optically diffuse (e.g., transluscent)materials. Also, it is generally desired that the support 21 isliquid-impermeable so that fluid flowing through the membrane 23 doesnot leak through the support 21. Examples of suitable materials for thesupport include, but are not limited to, glass; polymeric materials,such as polystyrene, polypropylene, polyester (e.g., Mylar® film),polybutadiene, polyvinylchloride, polyamide, polycarbonate, epoxides,methacrylates, and polymelamine; and so forth.

To provide a sufficient structural backing for the membrane 23, thesupport 21 is generally selected to have a certain minimum thickness.Likewise, the thickness of the support 21 is typically not so large asto adversely affect its optical properties. Thus, for example, thesupport 21 may have a thickness that ranges from about 100 to about5,000 micrometers, in some embodiments from about 150 to about 2,000micrometers, and in some embodiments, from about 250 to about 1,000micrometers. For instance, one suitable membrane strip having athickness of about 125 micrometers may be obtained from Millipore Corp.of Bedford, Mass. under the name “SHF180UB25.”

As is well known the art, the membrane 23 may be cast onto the support21, wherein the resulting laminate may be die-cut to the desired sizeand shape. Alternatively, the membrane 23 may simply be laminated to thesupport 21 with, for example, an adhesive. In some embodiments, anitrocellulose or nylon membrane is adhered to a Mylar® film. Anadhesive is used to bind the membrane to the Mylar® film, such as apressure-sensitive adhesive. Laminate structures of this type arebelieved to be commercially available from Millipore Corp. of Bedford,Mass. Still other examples of suitable laminate assay device structuresare described in U.S. Pat. No. 5,075,077 to Durley, Ill, et al., whichis incorporated herein in its entirety by reference thereto for allpurposes.

The device 20 may also contain an absorbent pad (not shown). Forexample, the absorbent pad may be positioned adjacent to or near an end27 of the membrane 23. The absorbent pad generally receives fluid thathas migrated through the entire membrane 23. The absorbent pad mayassist in promoting capillary action and fluid flow through the membrane23.

The test membrane 23 includes a collection region 68, which is a portionof the membrane having precisely defined dimensions. The collectionregion 68 collects and stores the test sample before the sample isconducted to a detection region 31. Upon saturation of the collectionregion 68, a precisely metered volume of the test sample is thusobtained. The correlation of amount of test sample at a saturationcondition of a given volume of the collection region 68 may beempirically determined for various test samples and membrane materialsand plotted or otherwise stored in a convenient format.

Referring to FIG. 1, the collection region 68 is separated from thedetection region 31 by a barrier 62. This barrier 62 serves to isolatethe collection region 68 and prevent migration of the test sample to thedetection region 31 until the collection region 68 has been saturatedwith the test sample. Once the collection region 68 has been saturated(and the required volume of test sample is thus obtained) the barrier 62is effectively “removed” so that the test sample can be conducted fromthe collection region 68 to the detection region 31 by application of adiluent to the collection region 68.

The barrier 62 may take on various forms. For example, in the embodimentillustrated in FIGS. 1, 2, and 6, the barrier 62 is defined by a solublecomposition 64 that is applied in a defined band or stripe across thewidth of the membrane 23. This composition 64 permeates through themembrane 23 to the support 21 to effectively define a “dam” across themembrane 23. The soluble composition 64 must thus have a viscosity topermeate through the membrane 23 and be carefully applied in a welldefined band across the membrane 23 so as not to compromise the leadingedge dimension of the collection region 68.

In a desirable embodiment, the soluble composition 64 is dissolvable bythe diluent that is used to wash the test sample from the collectionregion 68 to the detection region 31. The soluble composition isselected so as not to interfere with or mask the analytical testconducted on the test sample at the detection region 31. In particularembodiments, the soluble composition may be, for example, sugar,glycerol, and salts.

In other embodiments, for example as illustrated in FIGS. 3, 4, and 5,the barrier 62 is defined by physical structure that essentiallyseparates the collection region 68 from the downstream region of themembrane 23 containing the detection region 31. This structure may be,for example, a separation or divide 66 in the membrane 23 that spans thewidth of the membrane. This divide 66 prevents the test sample that hasaccumulated in the collection region 68 from migrating to the detectionregion 31. To initiate flow of the test sample from the collectionregion 68 to the detection zone 31, a variety of techniques may beemployed. For example, referring to FIG. 5, a bridging member 51 isshown that is placed over the divide 66 so that it is in fluidcommunication with the membrane 23. More specifically, the bridgingmember 51 has a first end 53 that is contiguous and in fluidcommunication with the membrane 23 at a location nearer to the detectionzone 31 and a second opposing end 55 that is also contiguous and influid communication with the collection region 68 of the membrane 23.The bridging member 51 provides a capillary “lift” that pulls the testsample volume and diluent from the collection region 68. Once absorbedby the bridging member 51, the test sample is capable of flowing alongthe bridging member 51 and through the membrane 23 to the detection zone31 for analysis. The bridging member 51 may be formed from any materialthrough which the test sample is capable of flowing. For example, thebridging member 51 may be formed from any of the membrane-basedmaterials described above for use in forming the membrane 23. Somespecific materials that may be used include, but are not limited to,nylon, nitrocellulose, cellulose, porous polyethylene pads, and glassfiber filter paper.

When blood is the test sample, the bridging member 51 may also serve thefunction of a blood separation filter. Blood separation filtersselectively retain cellular components (e.g., red blood cells) containedwithin a whole blood sample and deliver the remaining components of theblood sample (e.g., plasma or serum) to the detection zone. The bloodseparation filter may be made of any suitable material, for example, ahydrophobic material capable of filtering cells (e.g., blood cells) fromfluids. Various packings or sieving depth filters may be employed, suchas glass fibers, cellulose or glass filters treated with red blood cellcapture reagents, glass fiber filters, synthetic fiber filters or acomposite material including any combination of the above materials.Glass fiber filters, for instance, are commercially available fromWhatman plc of Kent, United Kingdom; Millipore Corp. of Billerica,Mass.; and Pall Corp. of Ann Arbor, Mich. Such glass fiber filters mayhave a fiber diameter in the range of about 0.05 to about 9 micrometersand a density of about 50 to about 150 g/m². Other examples of suitableblood separation filters are described in U.S. Pat. No. 5,416,000 toAllen, et al., as well as U.S. Patent Application Publication Nos.2004/0126833 to Shull, et al. and 2003/0032196 to Zhou, all of which areincorporated herein in their entirety by reference thereto for allpurposes. If desired, the blood separation filter may be treated withone or more reagents (e.g., agglutinin), such as described above.

The test sample may be applied to the collection region 68 by variousmeans. For example, the user may deposit the test sample directly ontothe collection region 68 by a pipette, dropper, or other device. Inalternate embodiments, the test sample may be initially received in achannel structure defined in the membrane 23 that aids in rapid intakeof the sample and uniform distribution of the sample across the width ofthe collection region 68. For example, referring again to FIG. 1, thelateral flow device 20 may include a channel 35 that is formed in asurface of the membrane 23 upstream of the collection region 68.Although illustrated as having a rectangular shape, the channel 35 maygenerally have any desired cross-sectional shape, such as circular,square, triangular, trapezoidal, v-shaped, u-shaped, hexagonal,octagonal, irregular, and so forth. Further, the channel 35 may bestraight, tapered, curved, serpentine, labyrinth-like, or have any otherdesired configuration.

Regardless of the shape selected, the dimensions of the channel 35 aregenerally such that it is capable of rapidly taking up the test samplevia passive capillary flow. Capillary flow generally occurs when theadhesive forces of a fluid to the walls of a channel are greater thanthe cohesive forces between the liquid molecules. Specifically,capillary pressure is inversely proportional to the cross-sectionaldimension of the channel and directly proportional to the surfacetension of the liquid, multiplied by the cosine of the contact angle ofthe fluid in contact with the material forming the channel. Thus, tofacilitate capillary flow, the length of the channel 35 in thelongitudinal direction “L” of the membrane 23 may be less than about 20millimeters, in some embodiments from about 0.001 to about 10millimeters, and in some embodiments, from about 0.01 to about 4millimeters. Of course, the length may also vary as a function of width.

The dimensions of the channel 35 dictate the volume of the test samplethat will be delivered to the collection zone 68. More specifically, thetest sample will quickly fill the empty volume of the channel 35 uponapplication thereto, thereby controlling the amount of sample deliveredto the device 20. In this regard, the channel 35 should have dimensionsto ensure that a sufficient volume of the sample is present for completesaturation of the collection region 68. To facilitate the delivery of acontrolled volume of the test sample to the collection zone 31, theheight or depth of the channel 35 may be varied to accommodate thedesired volume of the test sample. For example, the depth of the channel35 may be from about 0.1 micrometers to about 800 micrometers, in someembodiments from about 20 micrometers to about 400 micrometers, and insome embodiments, from about 80 micrometers to about 200 micrometers.The channel 35 may also have a width (in a direction “W”) that is thesame or substantially equal to the width of the membrane 23. In thismanner, the test sample will flow more uniformly across the entire widthof the collection region 68 upon initiation of the assay. In turn, thetest sample will ultimately reach the detection zone 31 in a moreuniform manner, thereby providing more accurate results. In someembodiments, for instance, the width of the metering channel 35 rangesfrom about 0.5 to about 20 millimeters, in some embodiments from about 1to about 15 millimeters, and in some embodiments, from about 2 to about10 millimeters. Of course, the width, depth, and/or length of thechannel 35 may also vary as a function of the dimension. In such cases,the specified width, depth, or length is an average dimension.

The ability of the channel 35 to take up an aqueous sample (e.g., blood)by capillary action is improved when its surface tension is near orexceeds the surface tension of water (i.e., 72 mN/m). Thus, if desired,the metering channel 35 may be treated with one or more wetting agentsto increase surface tension. One type of wetting agent that may be usedin the present invention is a hydrophilic wetting agent, such as anonionic surfactant. Examples of suitable nonionic surfactants includeethoxylated alkylphenols, ethoxylated and propoxylated fatty alcohols,ethylene oxide-propylene oxide block copolymers, ethoxylated esters offatty (C₈-C₁₈) acids, condensation products of ethylene oxide with longchain amines or amides, condensation products of ethylene oxide withalcohols, acetylenic diols, and mixtures thereof. Various specificexamples of suitable nonionic surfactants include, but are not limitedto, methyl gluceth-10, PEG-20 methyl glucose distearate, PEG-20 methylglucose sesquistearate, C₁₁₋₁₅ pareth-20, ceteth-8, ceteth-12,dodoxynol-12, laureth-15, PEG-20 castor oil, polysorbate 20,steareth-20, polyoxyethylene-10 cetyl ether, polyoxyethylene-10 stearylether, polyoxyethylene-20 cetyl ether, polyoxyethylene-10 oleyl ether,polyoxyethylene-20 oleyl ether, an ethoxylated nonylphenol, ethoxylatedoctylphenol, ethoxylated dodecylphenol, or ethoxylated fatty (C₆-C₂₂)alcohol, including 3 to 20 ethylene oxide moieties, polyoxyethylene-20isohexadecyl ether, polyoxyethylene-23 glycerol laurate,polyoxyethylene-20 glyceryl stearate, PPG-10 methyl glucose ether,PPG-20 methyl glucose ether, polyoxyethylene-20 sorbitan monoesters,polyoxyethylene-80 castor oil, polyoxyethylene-15 tridecyl ether,polyoxyethylene-6 tridecyl ether, laureth-2, laureth-3, laureth-4, PEG-3castor oil, PEG 600 dioleate, PEG 400 dioleate, and mixtures thereof.Commercially available nonionic surfactants may include the SURFYNOL®range of acetylenic diol surfactants available from Air Products andChemicals of Allentown, Pa.; the TWEEN® range of polyoxyethylenesurfactants available from Fisher Scientific of Pittsburgh, Pa.; and theTRITON® range of polyoxyethylene surfactants (e.g., TRITON® X-100,polyoxyethylene-10 isooctylcyclohexyl ether) available fromSigma-Aldrich Chemical Co. of St. Louis, Mo.

Besides surfactants, still other suitable wetting agents may includewater-soluble or water-swellable polymers that are substantially morelubricious when wetted with water, or with a water or alcohol-basedelectrolyte, than when dry. Examples of such hydrophilic polymersinclude, for instance, sodium, potassium and calcium alginates,carboxymethylcellulose, agar, gelatin, polyvinyl alcohol, collagen,pectin, chitin, chitosan, poly(α-amino acids), polyester,poly-1-caprolactone, polyvinylpyrrolidone, polyethylene oxide, polyvinylalcohol, polyether, polysaccharide, hydrophilic polyurethane,polyhydroxyacrylate, polymethacrylate, dextran, xanthan, hydroxypropylcellulose, methyl cellulose, and homopolymers and copolymers ofN-vinylpyrrolidone, N-vinyllactam, N-vinyl butyrolactam, N-vinylcaprolactam, other vinyl compounds having polar pendant groups, acrylateand methacrylate having hydrophilic esterifying groups, hydroxyacrylate,acrylic acid, and combinations thereof.

Referring again to FIG. 1, a score or cut line 70 is indicated by adashed line. This line may be desired to precisely define the dimensionsof the collection region 68 once the entire region of the membrane 23between the channel 35 and barrier 62 has been saturated with the testsample. This region may be considered as a “receiving region”, with thecollection region 68 being a sub-part of the receiving region that issubsequently defined along the line 70. Prior to supplying the diluentto the collection region 68, it may be necessary to separate thecollection region 68 from the channel 35, for example by cutting orscoring the membrane 23 along the line 70. This prevents any excess testsample within the channel 35 from being conveyed to the detection region31, which would alter the defined volume of the test sample conveyedfrom the collection region 68.

The channel 35 (and divide 66) may generally be formed using any of avariety of different techniques. For example, the channel 35 may beformed by simply laminating separate portions of a membrane onto asupport material so that a channel is formed therebetween. As a result,the walls of the metering channel 35 are at least partially formed byrespective membrane structures. The wall 38 of the channel 35 may beconductive to the test sample across the width of the channel 35 so thatthe test sample migrates into the “receiving” region of the membrane. Asdiscussed above, the collection region 68 may be separated from thereceiving region along line 70. In alternate embodiments, diffusion ofthe test sample from the channel 35 may be inhibited by treating one orboth of the walls with a hydrophobic material (e.g., polymer). Forexample, referring to the embodiments of FIGS. 3 and 4, the walls 38 and39 of the channel 35 are rendered non-conductive to the test sample(illustrated by the more dense region along the walls 38 and 39) toprevent the test sample from migrating from the channel 35 into thecollection region 68. In this case, a bridging member (such as member 51discussed above) may be used to conduct the test sample from the channel35 and into the collection region 68. The channel 35 has a bottomsurface defined by the support 21, which may be formed from a materialthat is hydrophobic.

In the embodiment of FIGS. 3 and 4, the non-conductive wall 38 of thechannel 35 prevents excess test sample from migrating into thecollection region 68 (after removal of the bridging member 51) and thusmay serve to adequately define the edge of the collection region 68 suchthat it is not necessary to separate the collection region 68 along aline 70.

The channel 35 (and divide 66) may be microfabricated into the membrane23. Such a microfabrication technique employs only a confined region ofthe membrane material for channel formation without adversely affectingthe remaining portions. Various mechanical microfabrication techniquesmay be used to accomplish such channel formation, and include, forinstance, cutting, laser ablation, photolithography, and so forth. Forexample, in one particular embodiment of the present invention, laserablation techniques are used to form the metering channel 35. Laserablation generally refers to a process for removing a material usingincident light of a certain wavelength. In polymeric materials, forinstance, the incident light generally induces photochemical changes inthe polymer that results in chemical dissolution. Any known laser may beemployed in the present invention, including, for instance, CO₂, pulsedlight lasers, diode lasers, ND Yag 1064 nm & 532 nm lasers, Alexandriteand Q-switched lasers, pulsed dye lasers, optical and RF lasers, erbiumlasers, ruby lasers, and holmium lasers. For example, a CO₂ laser may beused to etch a nitrocellulose membrane that is mounted on a supportingfixture. Through use of a moving beam or an X-Y table, precisionchannels may be generated on the nitrocellulose. In addition, variousother known optical devices may be employed in conjunction with thelaser to enhance the channel formation, such as optical lenses, mirrors,etc. The parameters of the laser ablation technique, such as wavelength,pulse duration, pulse repetition rate, and beam quality, may be selectedfor forming the channel 35 as is well known to those skilled in the art.

Chemical microfabrication techniques may also be employed in the presentinvention to form the channel 35 (and divide 66). For example, a solventtreatment may be employed in the present invention that exhibits adissolving capacity for the membrane 23. To ensure that dissolution ofthe membrane 23 remains confined within the regions of the channel 35,the dissolving capacity (solvency) of the solvent treatment is generallyoptimized so that it may quickly form the channel 35 before flowing toother regions of the membrane 23. Some examples of suitable solventsthat may be used in the solvent treatment include glycols, such aspropylene glycol, butylene glycol, triethylene glycol, hexylene glycol,polyethylene glycols, ethoxydiglycol, and dipropyleneglycol; glycolethers, such as methyl glycol ether, ethyl glycol ether, and isopropylglycol ether; ethers, such as diethyl ether and tetrahydrofuran;alcohols, such as methanol, ethanol, n-propanol, isopropanol, andbutanol; triglycerides; ketones, such as acetone, methyl ethyl ketone,and methyl isobutyl ketone; esters, such as ethyl acetate, butylacetate, and methoxypropyl acetate; amides, such as dimethylformamide,dimethylacetamide, dimethylcaprylic/capric fatty acid amide andN-alkylpyrrolidones; nitriles, such as acetonitrile, propionitrile,butyronitrile and benzonitrile; sulfoxides and sulfones, such asdimethyl sulfoxide (DMSO) and sulfolane; and so forth.

Of course, the selected solvent will vary depending on the material usedto form the membrane 23. In one particular embodiment, for example, themembrane 23 is formed from nitrocellulose. Examples of solvents that arecapable of dissolving nitrocellulose (i.e., active solvents) includeketones, such as acetone, methyl ethyl ketone, and methyl isobutylketone; esters, such as ethyl acetate, butyl acetate, and methoxy propylacetate; glycol ethers, such as methyl glycol ether, ethyl glycol ether,and isopropyl glycol ether; and alcohols, such as methanol and ethanol.In some embodiments, a latent solvent may be employed that is onlycapable of dissolving nitrocellulose under certain conditions, such asat a higher temperature or in the presence of an active solvent.Examples of such latent solvents may include, for instance, ethanol,isopropanol, and butanol. In some cases, a mixture of an active solventand a co-solvent (e.g., latent solvent or other active solvent) may beemployed. Such co-solvents may provide synergistic improvement to thedissolving capacity of the active solvent, or may simply be employed toreduce costs. When utilized, the active solvent is typically present inan amount greater than about 50 vol. %, in some embodiments greater thanabout 60 vol. %, and in some embodiments, from about 70 vol. % to about95 vol. %. Likewise, the co-solvent may be present in an amount lessthan about 50 vol. %, in some embodiments less than about 40 wt. %, andin some embodiments, from about 5 vol. % to about 30 vol. %. In stillother embodiments, a mixture of two or more latent solvents may beemployed.

The purity of a solvent may also influence its dissolving capacity. Thatis, higher solvent purities generally result in a higher dissolvingcapacity. Thus, to optimize dissolving capacity, it is normally desiredthat the purity of the solvent likewise be optimized. For example, inmost embodiments, the purity of a solvent employed in the presentinvention is greater than about 95 mass %, in some embodiments greaterthan about 98 mass %, and in some embodiments, greater than about 99mass %.

The solvent treatment may be applied to the membrane using any of avariety of well-known application techniques. Suitable applicationtechniques include, for example, spraying, printing (e.g., inkjet, pad,etc.), pipette, air brushing, metering with a dispensing pump, and soforth. In one particular embodiment, for example, the solvent treatmentis applied using a dispensing and optional drying process commonlyemployed to form detection lines on lateral flow strips. Such a systemcould involve placing a sheet of the porous membrane on a dispensingmachine and threading it through a rewind spindle. This may beaccomplished using either a batch or continuous process. The dispensingmachine delivers a precise volume of the solvent treatment in a straightline as the membrane passes beneath. The sheet then passes through adrier and is wound back on a spool for further processing. One suchlab-scale dispensing pump system for batch processes is available fromKinematic Automation, Inc. of Twain Harte, Calif. under the name“Matrix™ 1600.”

The solvent treatment may also be applied in any amount effective toform the channel 35 with the desired size and shape. The ultimate amountemployed may depend on a variety of factors, including the dissolvingcapacity of the solvent for the membrane 23, the speed of application,etc. For example, in some embodiments, the solvent treatment is appliedin an amount of from about 0.01 to about 10 microliters per centimeterin width of the membrane, in some embodiments from about 0.1 to about 10microliters per centimeter in width of the membrane, and in someembodiments, from about 0.5 to about 5 microliters per centimeter inwidth of the membrane 23.

One benefit of the above-described microfabrication techniques is thatthey may impart barrier properties to the walls of the channel 35 anddivide 66 without requiring separate treatment, such as with ahydrophobic material. For example, referring to FIG. 4, one embodimentof a channel 35 and divide 66 are shown in cross-section that have beensubjected to a microfabrication technique. Although the membrane 23 ofthis embodiment contains pores 60, any pores previously located near thewalls of the channel 35 and divide 66 are either destroyed orsubstantially reduced in size subsequent to the microfabricationtechnique. Likewise, the channel 35 and divide 66 have a bottom surface41 defined by the support 21, which is generally formed from a materialthat is hydrophobic. In this manner, flow of the test sample through thewalls and beneath the membrane 23 is substantially inhibited.Alternatively, the channel 35 and divide 66 may not extend to thesupport 21 so that the bottom surface of the structures is formed by themembrane 23. In such cases, the microfabrication technique may alsodestroy any pores previously located near the bottom surface.

Regardless of the manner in which it is formed, the channel 35 acts as amechanism for collecting the test sample until initiation of the assayis desired. The test sample may be applied directly to the meteringchannel 35 by a user, or it may be supplied to the metering channel 35from some other location of the assay device 20, such as from a samplepad, a blood filter, etc. In one embodiment, a user may simply apply adrop of whole blood (e.g., drop obtained from a lancet, a finger, orother alternate site, such as a forearm) to the channel 35.

If desired, the assay device 20 may be configured to facilitateapplication of the test sample to the channel 35. Referring to FIG. 6,for instance, one embodiment of the assay device 20 is shown thatincludes a channel 35, a membrane 23, and a support 21. In thisparticular embodiment, portions of the membrane 23 and support 21 areremoved so that a tip 24 is formed at the channel 35. For example, thetip 24 may provide a location against which a user's skin may bedepressed, thereby transferring blood to the metering channel 35.

When the test sample is whole blood, the metering channel 35 may betreated with a red blood cell agglutinating reagent (i.e., agglutinin)to facilitate separation of the red blood cells from the blood serum.For example, the walls 38, 39 and/or surface 41 of the channel 35 may bepretreated with such an agglutinating agent. In this manner, only theblood serum or plasma is analyzed at the detection zone 31, which mayenhance the semi-quantitative or quantitative detection of low volumetest samples. Agglutinin may be a lectin, such as concanavalin A orLycopersicon esculentum, or an antibody that specifically bindserythrocytes, such as a polyclonal rabbit anti-human erythrocyteantibody preparation. Agglutinins are typically applied in an amountsufficient to agglutinate most of the erythrocytes in the test sample.Other reagents may also be applied to selectively bind or retardmovement of certain other biological sample constituents. For example,the metering channel 35 may be treated with a reagent that separates redblood cells from plasma so that plasma components, such as an analyte(e.g., C-reactive protein), may be analyzed. Alternatively, a reagentmay be applied that selectively separates biological sample componentsby their biological, chemical, or physical properties. Other reagentsthat reduce non-specific binding or non-specific adsorption ofcomponents of the blood sample may be used to treat the channel 35. Forinstance, the channel 35 may be treated with a protein, such as albumin(e.g., bovine serum albumin).

To further aid in transfer of the test sample from the channel 35 to thecollection region 68, an absorbent member may be placed into the channel35 to absorb the test sample. The bridging member 51 is then placedcontiguous and in fluid communication with the absorbent member. In thismanner, the test sample may simply flow from the absorbent member to thebridging member 51.

Regardless of the particular mechanism or method used to saturate thecollection region 68 with the test sample, a diluent (or washing agent)is generally employed to facilitate delivery of the test sample from thecollection region 68 to the detection zone 31. The diluent is typicallyapplied upstream from the collection region 68 so that it may initiateflow in the direction of the detection zone 31. For example, in theembodiment of FIG. 5, upon application, the diluent will mix with thetest sample and flow through the membrane 23 of the collection region 68until reaching the first end 53 of the bridging member 51. The diluentand test sample will then flow through the bridging member 51 to thesecond end 55 of the bridging member 51. Finally, the diluent/testsample mixture flows from the second end 55 to the detection zone 31 foranalysis.

In the embodiment of FIGS. 1 and 2, diluent/test sample mixture flowsthrough the membrane 23 of the collection region 68 until encounteringthe soluble composition 64. The diluent will dissolve the composition 64and the mixture is free to flow into the detection zone 31.

The diluent may be any material having a viscosity that is sufficientlylow to allow movement of the fluid by capillary action and that supportsa reaction between the analyte and any binding agents (e.g., does notinterfere with antibody/antigen interaction). In one embodiment, thediluent contains water, a buffering agent; a salt (e.g., NaCl); aprotein stabilizer (e.g., BSA, casein, trehalose, or serum); and/or adetergent (e.g., nonionic surfactant). Representative buffering agentsinclude, for example, phosphate-buffered saline (PBS) (e.g., pH of 7.2),2-(N-morpholino)ethane sulfonic acid (MES) (e.g., pH of 5.3), HEPESbuffer, TBS buffer, etc., and so forth.

In addition to the components set forth above, the diagnostic test kitof the present invention may also contain various other components toenhance detection accuracy. For exemplary purposes only, one embodimentof an immunoassay that may be performed in accordance with the presentinvention to detect the presence will now be described in more detail.Immunoassays utilize mechanisms of the immune systems, whereinantibodies are produced in response to the presence of antigens that arepathogenic or foreign to the organisms. These antibodies and antigens,i.e., immunoreactants, are capable of binding with one another, therebycausing a highly specific reaction mechanism that may be used todetermine the presence or concentration of that particular antigen in abiological sample.

To initiate the immunoassay, the test sample (e.g., whole blood) isinitially applied to the channel 35 or directly onto the collectionregion 68, such as with a lancet, needle, dropper, pipette, capillarydevice, etc. Once the collection region 68 is saturated with the testsample such that a known desired volume of the test sample is containedwithin the collection region 68, the bridging member 51 is placed overthe divide 66 in the embodiment of FIGS. 4 and 5 and the diluent isapplied to the device 20. The application of the bridging member 51 anddiluent may occur simultaneously or sequentially, and may be performedby manual or automatic operation. The location of diluent applicationmay vary as desired. For example, in some embodiments, the diluent isapplied to an additional membrane, such as a sample pad (not shown) orconjugate pad (not shown), which are in fluid communication with amembrane 23. The sample pad and conjugate pad may be formed from anymaterial through which a fluid is capable of passing, such as glassfibers. Further, if desired, the channel 35 may be formed in the samplepad and/or conjugate pad in the manner described above.

To facilitate the detection of the analyte within the test sample, asubstance may be pre-applied to the sample pad and/or conjugate pad, orpreviously mixed with the diluent or test sample, which is detectableeither visually or by an instrumental device. Any substance generallycapable of producing a signal that is detectable visually or by aninstrumental device may be used as detection probes. Suitable detectablesubstances may include, for instance, luminescent compounds (e.g.,fluorescent, phosphorescent, etc.); radioactive compounds; visualcompounds (e.g., colored dye or metallic substance, such as gold);liposomes or other vesicles containing signal-producing substances;enzymes and/or substrates, and so forth. Other suitable detectablesubstances may be described in U.S. Pat. No. 5,670,381 to Jou, et al.and U.S. Pat. No. 5,252,459 to Tarcha, et al., which are incorporatedherein in their entirety by reference thereto for all purposes. If thedetectable substance is colored, the ideal electromagnetic radiation islight of a complementary wavelength. For instance, blue detection probesstrongly absorb red light.

In some embodiments, the detectable substance may be a luminescentcompound that produces an optically detectable signal. For example,suitable fluorescent molecules may include, but are not limited to,fluorescein, europium chelates, phycobiliprotein, rhodamine, and theirderivatives and analogs. Other suitable fluorescent compounds aresemiconductor nanocrystals commonly referred to as “quantum dots.” Forexample, such nanocrystals may contain a core of the formula CdX,wherein X is Se, Te, S, and so forth. The nanocrystals may also bepassivated with an overlying shell of the formula YZ, wherein Y is Cd orZn, and Z is S or Se. Other examples of suitable semiconductornanocrystals may also be described in U.S. Pat. No. 6,261,779 toBarbera-Guillem, et al. and U.S. Pat. No. 6,585,939 to Dapprich, whichare incorporated herein in their entirety by reference thereto for allpurposes.

Further, suitable phosphorescent compounds may include metal complexesof one or more metals, such as ruthenium, osmium, rhenium, iridium,rhodium, platinum, indium, palladium, molybdenum, technetium, copper,iron, chromium, tungsten, zinc, and so forth. Especially preferred areruthenium, rhenium, osmium, platinum, and palladium. The metal complexmay contain one or more ligands that facilitate the solubility of thecomplex in an aqueous or non-aqueous environment. For example, somesuitable examples of ligands include, but are not limited to, pyridine;pyrazine; isonicotinamide; imidazole; bipyridine; terpyridine;phenanthroline; dipyridophenazine; porphyrin, porphine, and derivativesthereof. Such ligands may be, for instance, substituted with alkyl,substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl,carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxy, imino,hydroxycarbonyl, aminocarbonyl, amidine, guanidinium, ureide,sulfur-containing groups, phosphorus containing groups, and thecarboxylate ester of N-hydroxy-succinimide.

Porphyrins and porphine metal complexes possess pyrrole groups coupledtogether with methylene bridges to form cyclic structures with metalchelating inner cavities. Many of these molecules exhibit strongphosphorescence properties at room temperature in suitable solvents(e.g., water) and an oxygen-free environment. Some suitable porphyrincomplexes that are capable of exhibiting phosphorescent propertiesinclude, but are not limited to, platinum (II) coproporphyrin-I and III,palladium (II) coproporphyrin, ruthenium coproporphyrin,zinc(II)-coproporphyrin-I, derivatives thereof, and so forth. Similarly,some suitable porphine complexes that are capable of exhibitingphosphorescent properties include, but not limited to, platinum(II)tetra-meso-fluorophenylporphine and palladium(II)tetra-meso-fluorophenylporphine. Still other suitable porphyrin and/orporphine complexes are described in U.S. Pat. No. 4,614,723 to Schmidt,et al.; U.S. Pat. No. 5,464,741 to Hendrix; U.S. Pat. No. 5,518,883 toSoini; U.S. Pat. No. 5,922,537 to Ewart, et al.; U.S. Pat. No. 6,004,530to Sagner, et al.; and U.S. Pat. No. 6,582,930 to Ponomarev, et al.,which are incorporated herein in their entirety by reference thereto forall purposes.

Bipyridine metal complexes may also be utilized as phosphorescentcompounds. Some examples of suitable bipyridine complexes include, butare note limited to,bis[(4,4′-carbomethoxy)-2,2′-bipyridine]2-[3-(4-methyl-2,2′-bipyridine-4-yl)propyl]-1,3-dioxolaneruthenium (II);bis(2,2′bipyridine)[4-(butan-1-al)-4′-methyl-2,2′-bi-pyridine]ruthenium(II); bis(2,2′-bipyridine)[4-(4′-methyl-2,2′-bipyridine-4′-yl)-butyricacid]ruthenium (II); tris(2,2′bipyridine)ruthenium (II);(2,2′-bipyridine)[bis-bis(1,2-diphenylphosphino)ethylene]2-[3-(4-methyl-2,2′-bipyridine-4′-yl)propyl]-1,3-dioxolaneosmium (II);bis(2,2′-bipyridine)[4-(4′-methyl-2,2′-bipyridine)-butylamine]ruthenium(II);bis(2,2′-bipyridine)[1-bromo-4(4′-methyl-2,2′-bipyridine-4-yl)butane]ruthenium(II); bis(2,2′-bipyridine)maleimidohexanoic acid,4-methyl-2,2′-bipyridine-4′-butylamide ruthenium (II), and so forth.Still other suitable metal complexes that may exhibit phosphorescentproperties may be described in U.S. Pat. No. 6,613,583 to Richter, etal.; U.S. Pat. No. 6,468,741 to Massey, et al.; U.S. Pat. No. 6,444,423to Meade, et al.; U.S. Pat. No. 6,362,011 to Massey, et al.; U.S. Pat.No. 5,731,147 to Bard, et al.; and U.S. Pat. No. 5,591,581 to Massey, etal., which are incorporated herein in their entirety by referencethereto for all purposes.

In some cases, luminescent compounds may have a relatively long emissionlifetime may have a relatively large “Stokes shift.” The term “Stokesshift” is generally defined as the displacement of spectral lines orbands of luminescent radiation to a longer emission wavelength than theexcitation lines or bands. A relatively large Stokes shift allows theexcitation wavelength of a luminescent compound to remain far apart fromits emission wavelengths and is desirable because a large differencebetween excitation and emission wavelengths makes it easier to eliminatethe reflected excitation radiation from the emitted signal. Further, alarge Stokes shift also minimizes interference from luminescentmolecules in the sample and/or light scattering due to proteins orcolloids, which are present with some body fluids (e.g., blood). Inaddition, a large Stokes shift also minimizes the requirement forexpensive, high-precision filters to eliminate background interference.For example, in some embodiments, the luminescent compounds have aStokes shift of greater than about 50 nanometers, in some embodimentsgreater than about 100 nanometers, and in some embodiments, from about100 to about 350 nanometers.

For example, exemplary fluorescent compounds having a large Stokes shiftinclude lanthanide chelates of samarium (Sm (III)), dysprosium (Dy(III)), europium (Eu (III)), and terbium (Tb (III)). Such chelates mayexhibit strongly red-shifted, narrow-band, long-lived emission afterexcitation of the chelate at substantially shorter wavelengths.Typically, the chelate possesses a strong ultraviolet excitation banddue to a chromophore located close to the lanthanide in the molecule.Subsequent to excitation by the chromophore, the excitation energy maybe transferred from the excited chromophore to the lanthanide. This isfollowed by a fluorescence emission characteristic of the lanthanide.Europium chelates, for instance, have Stokes shifts of about 250 toabout 350 nanometers, as compared to only about 28 nanometers forfluorescein. Also, the fluorescence of europium chelates is long-lived,with lifetimes of about 100 to about 1000 microseconds, as compared toabout 1 to about 100 nanoseconds for other fluorescent labels. Inaddition, these chelates have a narrow emission spectra, typicallyhaving bandwidths less than about 10 nanometers at about 50% emission.One suitable europium chelate is N-(p-isothiocyanatobenzyl)-diethylenetriamine tetraacetic acid-Eu⁺³.

In addition, lanthanide chelates that are inert, stable, andintrinsically fluorescent in aqueous solutions or suspensions may alsobe used in the present invention to negate the need for micelle-formingreagents, which are often used to protect chelates having limitedsolubility and quenching problems in aqueous solutions or suspensions.One example of such a chelate is4-[2-(4-isothiocyanatophenyl)ethynyl]-2,6-bis([N,N-bis(carboxymethyl)amino]methyl)-pyridine[Ref: Lovgren, T., et al.; Clin. Chem. 42, 1196-1201 (1996)]. Severallanthanide chelates also show exceptionally high signal-to-noise ratios.For example, one such chelate is a tetradentate β-diketonate-europiumchelate [Ref: Yuan, J. and Matsumoto, K.; Anal. Chem. 70, 596-601(1998)]. In addition to the fluorescent labels described above, otherlabels that are suitable for use in the present invention may bedescribed in U.S. Pat. No. 6,030,840 to Mullinax, et al.; U.S. Pat. No.5,585,279 to Davidson; U.S. Pat. No. 5,573,909 to Singer, et al.; U.S.Pat. No. 6,242,268 to Wieder, et al.; and U.S. Pat. No. 5,637,509 toHemmila, et al., which are incorporated herein in their entirety byreference thereto for all purposes.

Detectable substances, such as described above, may be used alone or inconjunction with a particle (sometimes referred to as “beads” or“microbeads”). For instance, naturally occurring particles, such asnuclei, mycoplasma, plasmids, plastids, mammalian cells (e.g.,erythrocyte ghosts), unicellular microorganisms (e.g., bacteria),polysaccharides (e.g., agarose), etc., may be used. Further, syntheticparticles may also be utilized. For example, in one embodiment, latexmicroparticles that are labeled with a fluorescent or colored dye areutilized. Although any synthetic particle may be used in the presentinvention, the particles are typically formed from polystyrene,butadiene styrenes, styreneacrylic-vinyl terpolymer,polymethylmethacrylate, polyethylmethacrylate, styrene-maleic anhydridecopolymer, polyvinyl acetate, polyvinylpyridine, polydivinylbenzene,polybutyleneterephthalate, acrylonitrile, vinylchloride-acrylates, andso forth, or an aldehyde, carboxyl, amino, hydroxyl, or hydrazidederivative thereof. Other suitable particles may be described in U.S.Pat. No. 5,670,381 to Jou, et al.; U.S. Pat. No. 5,252,459 to Tarcha, etal.; and U.S. Patent Publication No. 2003/0139886 to Bodzin, et al.,which are incorporated herein in their entirety by reference thereto forall purposes. Commercially available examples of suitable fluorescentparticles include fluorescent carboxylated microspheres sold byMolecular Probes, Inc. under the trade names “FluoSphere” (Red 580/605)and “TransfluoSphere” (543/620), as well as “Texas Red” and 5- and6-carboxytetramethylrhodamine, which are also sold by Molecular Probes,Inc. In addition, commercially available examples of suitable colored,latex microparticles include carboxylated latex beads sold by Bang'sLaboratory, Inc. Metallic particles (e.g., gold particles) may also beutilized in the present invention.

When utilized, the shape of the particles may generally vary. In oneparticular embodiment, for instance, the particles are spherical inshape. However, it should be understood that other shapes are alsocontemplated by the present invention, such as plates, rods, discs,bars, tubes, irregular shapes, etc. In addition, the size of theparticles may also vary. For instance, the average size (e.g., diameter)of the particles may range from about 0.1 nanometers to about 100microns, in some embodiments, from about 1 nanometer to about 10microns, and in some embodiments, from about 10 to about 100 nanometers.

In some instances, it may be desired to modify the detection probes insome manner so that they are more readily able to bind to the analyte.In such instances, the detection probes may be modified with certainspecific binding members that are adhered thereto to form conjugatedprobes. Specific binding members generally refer to a member of aspecific binding pair, i.e., two different molecules where one of themolecules chemically and/or physically binds to the second molecule. Forinstance, immunoreactive specific binding members may include antigens,haptens, aptamers, antibodies (primary or secondary), and complexesthereof, including those formed by recombinant DNA methods or peptidesynthesis. An antibody may be a monoclonal or polyclonal antibody, arecombinant protein or a mixture(s) or fragment(s) thereof, as well as amixture of an antibody and other specific binding members. The detailsof the preparation of such antibodies and their suitability for use asspecific binding members are well known to those skilled in the art.Other common specific binding pairs include but are not limited to,biotin and avidin (or derivatives thereof), biotin and streptavidin,carbohydrates and lectins, complementary nucleotide sequences (includingprobe and capture nucleic acid sequences used in DNA hybridizationassays to detect a target nucleic acid sequence), complementary peptidesequences including those formed by recombinant methods, effector andreceptor molecules, hormone and hormone binding protein, enzymecofactors and enzymes, enzyme inhibitors and enzymes, and so forth.Furthermore, specific binding pairs may include members that are analogsof the original specific binding member. For example, a derivative orfragment of the analyte (i.e., “analog”) may be used so long as it hasat least one epitope in common with the analyte.

The specific binding members may generally be attached to the detectionprobes using any of a variety of well-known techniques. For instance,covalent attachment of the specific binding members to the detectionprobes (e.g., particles) may be accomplished using carboxylic, amino,aldehyde, bromoacetyl, iodoacetyl, thiol, epoxy and other reactive orlinking functional groups, as well as residual free radicals and radicalcations, through which a protein coupling reaction may be accomplished.A surface functional group may also be incorporated as a functionalizedco-monomer because the surface of the detection probe may contain arelatively high surface concentration of polar groups. In addition,although detection probes are often functionalized after synthesis, suchas with poly(thiophenol), the detection probes may be capable of directcovalent linking with a protein without the need for furthermodification. For example, in one embodiment, the first step ofconjugation is activation of carboxylic groups on the probe surfaceusing carbodiimide. In the second step, the activated carboxylic acidgroups are reacted with an amino group of an antibody to form an amidebond. The activation and/or antibody coupling may occur in a buffer,such as phosphate-buffered saline (PBS) (e.g., pH of 7.2) or2-(N-morpholino)ethane sulfonic acid (MES) (e.g., pH of 5.3). Theresulting detection probes may then be contacted with ethanolamine, forinstance, to block any remaining activated sites. Overall, this processforms a conjugated detection probe, where the antibody is covalentlyattached to the probe. Besides covalent bonding, other attachmenttechniques, such as physical adsorption, may also be utilized in thepresent invention.

Referring again to the figures in general, after passing through thecollection region 68, the diluent and test sample travel through themembrane 23 until reaching the detection zone 31. Upon reaching thedetection zone 31, the volume of the test sample is relatively uniformacross the entire width of the detection zone 31. In addition, as aresult of the known saturation volume of the collection region 68, thevolume of the test sample is also predetermined within a narrow range.

Within the detection zone 31, a receptive material is immobilized thatis capable of binding to the conjugated detection probes. The receptivematerial may be selected from the same materials as the specific bindingmembers described above, including, for instance, antigens; haptens;antibody-binding proteins, such as protein A, protein G, or protein A/G;neutravidin (a deglycosylated avidin derivative), avidin (a highlycationic 66,000-dalton glycoprotein), streptavidin (a nonglycosylated52,800-dalton protein), or captavidin (a nitrated avidin derivative);primary or secondary antibodies, and derivatives or fragments thereof.In one embodiment, for example, the receptive material is an antibodyspecific to an antigen within the test sample. The receptive materialserves as a stationary binding site for complexes formed between theanalyte and the conjugated detection probes. Specifically, analytes,such as antibodies, antigens, etc., typically have two or more bindingsites (e.g., epitopes). Upon reaching the detection zone 31, one ofthese binding sites is occupied by the specific binding member of theconjugated probe. However, the free binding site of the analyte may bindto the immobilized first receptive material. Upon being bound to theimmobilized receptive material, the complexed probes form a new ternarysandwich complex.

Other than the detection zone 31, the lateral flow device 20 may alsodefine various other zones for enhancing detection accuracy. Forexample, in embodiments in which high analyte concentrations are aconcern, the assay device 20 may contain an indicator zone 33 that ispositioned downstream from the detection zone 31 and is configured toprovide information as to whether the analyte concentration has reachedthe saturation concentration (“hook effect” region) for the assay. Theindicator zone 33 contains a second receptive material that isimmobilized on the membrane 23 and serves as a stationary binding sitefor the conjugated detection probes. To accomplish the desired bindingwithin the indicator zone 33, it is generally desired that the secondreceptive material is capable of differentiating between those detectionprobes that are complexed with the analyte and those that remainuncomplexed. For example, in one embodiment, the second receptivematerial includes a molecule that has at least one epitope in commonwith the analyte, such as analyte molecules, or derivatives or fragments(i.e., analog) thereof, so that it is capable of specifically binding toan antibody conjugate when it is uncomplexed with the analyte.

Alternatively, the second receptive material may include a biologicalmaterial that is not an analyte molecule or analog thereof, butnevertheless is capable of preferentially binding to uncomplexedconjugated detection probes. In one embodiment, for example, the firstreceptive material may be a monoclonal antibody, such as anti-CRP IgG₁.The detection probes are conjugated with a monoclonal antibody differentthan the monoclonal antibody of the first receptive material, such asanti-CRP IgG₂. In this particular embodiment, the second receptivematerial may be a secondary antibody, such as Goat anti-human, IgGF(ab′)₂, which has been adsorbed against F_(c) fragments and thereforereacts only with the F_(ab) portion of IgG. Thus, when no analyte ispresent, the secondary antibody is able to bind to the free “F_(ab)”binding domain of the anti-CRP IgG₂ monoclonal antibody. However, whenan antigen is present in the test sample, it first complexes with the“F_(ab)” binding domain of the anti-CRP IgG₂ monoclonal antibody. Thepresence of the antigen renders the “F_(ab)” binding domain unavailablefor subsequent binding with the secondary antibody. In this manner, thesecondary antibody within the indicator zone 35 is capable ofpreferentially binding to uncomplexed detection probes.

Although the detection zone 31 and optional indicator zone 33 mayprovide accurate results, it is sometimes difficult to determine therelative concentration of the analyte within the test sample underactual test conditions. Thus, the assay device 20 may include acalibration zone 32. In this embodiment, the calibration zone 32 isformed on the membrane 23 and is positioned downstream from thedetection zone 31 and optional indicator zone 35. Alternatively,however, the calibration zone 32 may also be positioned upstream fromthe detection zone 31 and/or optional indicator zone 33. The calibrationzone 32 is provided with a third receptive material that is capable ofbinding to any calibration probes that pass through the length of themembrane 23. When utilized, the calibration probes may contain adetectable substance that is the same or different than the detectablesubstance used for the detection probes. Moreover, the calibrationprobes may also be conjugated with a specific binding member, such asdescribed above. For example, in one embodiment, biotinylatedcalibration probes may be used. Generally speaking, the calibrationprobes are selected in such a manner that they do not bind to the firstor second receptive material at the detection zone 31 and indicator zone33. The third receptive material of the calibration zone 32 may be thesame or different than the receptive materials used in the detectionzone 31 or indicator zone 33. For example, in one embodiment, the thirdreceptive material is a biological receptive material, such as antigens,haptens, antibody-binding proteins (e.g., protein A, protein G, orprotein A/G), neutravidin, avidin, streptavidin, captavidin, primary orsecondary antibodies, or complexes thereof. It may also be desired toutilize various non-biological materials for the third receptivematerial (e.g., polyelectrolytes) of the calibration zone 32, such asdescribed in U.S. Patent Application Publication No. 2003/0124739 toSong, et al., which is incorporated herein in its entirety by referencethereto for all purposes.

When utilized, the polyelectrolytes may have a net positive or negativecharge, as well as a net charge that is generally neutral. For instance,some suitable examples of polyelectrolytes having a net positive chargeinclude, but are not limited to, polylysine (commercially available fromSigma-Aldrich Chemical Co., Inc. of St. Louis, Mo.), polyethyleneimine;epichlorohydrin-functionalized polyamines and/or polyamidoamines, suchas poly(dimethylamine-co-epichlorohydrin); polydiallyldimethyl-ammoniumchloride; cationic cellulose derivatives, such as cellulose copolymersor cellulose derivatives grafted with a quaternary ammoniumwater-soluble monomer; and so forth. In one particular embodiment,CelQuat® SC-230M or H-100 (available from National Starch & Chemical,Inc.), which are cellulosic derivatives containing a quaternary ammoniumwater-soluble monomer, may be utilized. Moreover, some suitable examplesof polyelectrolytes having a net negative charge include, but are notlimited to, polyacrylic acids, such as poly(ethylene-co-methacrylicacid, sodium salt), and so forth. It should also be understood thatother polyelectrolytes may also be utilized, such as amphiphilicpolyelectrolytes (i.e., having polar and non-polar portions). Forinstance, some examples of suitable amphiphilic polyelectrolytesinclude, but are not limited to, poly(styryl-b-N-methyl 2-vinylpyridnium iodide) and poly(styryl-b-acrylic acid), both of which areavailable from Polymer Source, Inc. of Dorval, Canada.

Although any polyelectrolyte may generally be used, the polyelectrolyteselected for a particular application may vary depending on the natureof the detection probes, the calibration probes, the membrane, and soforth. In particular, the distributed charge of a polyelectrolyte allowsit to bind to substances having an opposite charge. Thus, for example,polyelectrolytes having a net positive charge are often better equippedto bind with probes that are negatively charged, while polyelectrolytesthat have a net negative charge are often better equipped to bind toprobes that are positively charged. Thus, in such instances, the ionicinteraction between these molecules allows the required binding to occurwithin the calibration zone 32. Nevertheless, although ionic interactionis primarily utilized to achieve the desired binding in the calibrationzone 32, polyelectrolytes may also bind with probes having a similarcharge.

Because the polyelectrolyte is designed to bind to probes, it istypically desired that the polyelectrolyte be substantiallynon-diffusively immobilized on the surface of the membrane 23.Otherwise, the probes would not be readily detectable by a user. Thus,the polyelectrolytes may be applied to the membrane 23 in such a mannerthat they do not substantially diffuse into the matrix of the membrane23. In particular, the polyelectrolytes typically form an ionic and/orcovalent bond with functional groups present on the surface of themembrane 23 so that they remain immobilized thereon. Although notrequired, the formation of covalent bonds between the polyelectrolyteand the membrane 23 may be desired to more permanently immobilize thepolyelectrolyte thereon. For example, in one embodiment, the monomersused to form the polyelectrolyte are first formed into a solution andthen applied directly to the membrane 23. Various solvents (e.g.,organic solvents, water, etc.) may be utilized to form the solution.Once applied, the polymerization of the monomers is initiated usingheat, electron beam radiation, free radical polymerization, and soforth. In some instances, as the monomers polymerize, they form covalentbonds with certain functional groups of the membrane 23, therebyimmobilizing the resulting polyelectrolyte thereon. For example, in oneembodiment, an ethyleneimine monomer may form a covalent bond with acarboxyl group present on the surface of some membranes (e.g.,nitrocellulose).

In another embodiment, the polyelectrolyte may be formed prior toapplication to the membrane 23. If desired, the polyelectrolyte mayfirst be formed into a solution using organic solvents, water, and soforth. Thereafter, the polyelectrolytic solution is applied directly tothe membrane 23 and then dried. Upon drying, the polyelectrolyte mayform an ionic bond with certain functional groups present on the surfaceof the membrane 23 that have a charge opposite to the polyelectrolyte.For example, in one embodiment, positively-charged polyethyleneimine mayform an ionic bond with negatively-charged carboxyl groups present onthe surface of some membranes (e.g., nitrocellulose).

In addition, the polyelectrolyte may also be crosslinked to the membrane23 using various well-known techniques. For example, in someembodiments, epichlorohydrin-functionalized polyamines and/orpolyamidoamines may be used as a crosslinkable, positively-chargedpolyelectrolyte. Examples of these materials are described in U.S. Pat.No. 3,700,623 to Keim and U.S. Pat. No. 3,772,076 to Keim, U.S. Pat. No.4,537,657 to Keim, which are incorporated herein in their entirety byreference thereto for all purposes and are believed to be sold byHercules, Inc., Wilmington, Del. under the Kymene™ trade designation.For instance, Kymene™ 450 and 2064 are epichlorohydrin-functionalizedpolyamine and/or polyamidoamine compounds that contain epoxide rings andquaternary ammonium groups that may form covalent bonds with carboxylgroups present on certain types of membranes (e.g., nitrocellulose) andcrosslink with the polymer backbone of the membrane when cured. In someembodiments, the crosslinking temperature may range from about 50° C. toabout 120° C. and the crosslinking time may range from about 10 to about600 seconds.

Although various techniques for non-diffusively immobilizingpolyelectrolytes on the membrane 23 have been described above, it shouldbe understood that any other technique for non-diffusively immobilizingpolyelectrolytic compounds may be used in the present invention. Infact, the aforementioned methods are only intended to be exemplary ofthe techniques that may be used in the present invention. For example,in some embodiments, certain components may be added to thepolyelectrolyte solution that may substantially inhibit the diffusion ofsuch polyelectrolytes into the matrix of the membrane 23.

The detection zone 31, indicator zone 33, and calibration zone 32 mayeach provide any number of distinct detection regions so that a user maybetter determine the concentration of one or more analytes within a testsample. Each region may contain the same receptive materials, or maycontain different receptive materials. For example, the zones mayinclude two or more distinct regions (e.g., lines, dots, etc.). Theregions may be disposed in the form of lines in a direction that issubstantially perpendicular to the flow of the test sample through theassay device 20. Likewise, in some embodiments, the regions may bedisposed in the form of lines in a direction that is substantiallyparallel to the flow of the test sample through the assay device 20.

In some cases, the membrane 23 may also define a control zone (notshown) that gives a signal to the user that the assay is performingproperly. For instance, the control zone (not shown) may contain animmobilized receptive material that is generally capable of forming achemical and/or physical bond with probes or with the receptive materialimmobilized on the probes. Some examples of such receptive materialsinclude, but are not limited to, antigens, haptens, antibodies, proteinA or G, avidin, streptavidin, secondary antibodies, and complexesthereof. In addition, it may also be desired to utilize variousnon-biological materials for the control zone receptive material. Forinstance, in some embodiments, the control zone receptive material mayalso include a polyelectrolyte, such as described above, that may bindto uncaptured probes. Because the receptive material at the control zoneis only specific for probes, a signal forms regardless of whether theanalyte is present. The control zone may be positioned at any locationalong the membrane 23, but is preferably positioned downstream from thedetection zone 31 and the indicator zone 33.

Qualitative, semi-quantitative, and quantitative results may be obtainedin accordance with the present invention. For example, when it isdesired to semi-quantitatively or quantitatively detect an analyte, theintensity of any signals produced at the detection zone 31, indicatorzone 33, and/or calibration zone 32 may be measured with an opticalreader. The actual configuration and structure of the optical reader maygenerally vary as is readily understood by those skilled in the art. Forexample, optical detection techniques that may be utilized include, butare not limited to, luminescence (e.g., fluorescence, phosphorescence,etc.), absorbance (e.g., fluorescent or non-fluorescent), diffraction,etc. One suitable reflectance spectrophotometer is described, forinstance, in U.S. Patent App. Pub. No. 2003/0119202 to Kaylor, et al.,which is incorporated herein in its entirety by reference thereto forall purposes. In another embodiment, a reflectance-modespectrofluorometer may be used to detect the intensity of a fluorescencesignal. Suitable spectrofluorometers and related detection techniquesare described, for instance, in U.S. Patent App. Pub. No. 2004/0043502to Song, et al., which is incorporated herein in its entirety byreference thereto for all purposes. Likewise, a transmission-modedetection system may also be used to signal intensity.

Although various embodiments of device configurations have beendescribed above, it should be understood, that a device of the presentinvention may generally have any configuration desired, and need notcontain all of the components described above. Various other deviceconfigurations, for instance, are described in U.S. Pat. No. 5,395,754to Lambotte, et al.; U.S. Pat. No. 5,670,381 to Jou, et al.; and U.S.Pat. No. 6,194,220 to Malick, et al., which are incorporated herein intheir entirety by reference thereto for all purposes.

Various assay formats may also be used to test for the presence orabsence of an analyte using the assay device of the present invention.For instance, a “sandwich” format typically involves mixing the testsample with detection probes conjugated with a specific binding member(e.g., antibody) for the analyte to form complexes between the analyteand the conjugated probes. These complexes are then allowed to contact areceptive material (e.g., antibodies) immobilized within the detectionzone. Binding occurs between the analyte/probe conjugate complexes andthe immobilized receptive material, thereby localizing “sandwich”complexes that are detectable to indicate the presence of the analyte.This technique may be used to obtain quantitative or semi-quantitativeresults. Some examples of such sandwich-type assays are described byU.S. Pat. No. 4,168,146 to Grubb, et al. and U.S. Pat. No. 4,366,241 toTom, et al., which are incorporated herein in their entirety byreference thereto for all purposes. In a competitive assay, the labeledprobe is generally conjugated with a molecule that is identical to, oran analog of, the analyte. Thus, the labeled probe competes with theanalyte of interest for the available receptive material. Competitiveassays are typically used for detection of analytes such as haptens,each hapten being monovalent and capable of binding only one antibodymolecule. Examples of competitive immunoassay devices are described inU.S. Pat. No. 4,235,601 to Deutsch, et al., U.S. Pat. No. 4,442,204 toLiotta, and U.S. Pat. No. 5,208,535 to Buechler, et al., which areincorporated herein in their entirety by reference thereto for allpurposes. Various other device configurations and/or assay formats arealso described in U.S. Pat. No. 5,395,754 to Lambotte, et al.; U.S. Pat.No. 5,670,381 to Jou, et al.; and U.S. Pat. No. 6,194,220 to Malick, etal., which are incorporated herein in their entirety by referencethereto for all purposes.

As a result of the present invention, a controlled volume of a testsample may be uniformly delivered to a detection zone of a lateral flowassay device. Such control over sample flow provides a significantimprovement in detection accuracy and sensitivity for lateral flowsystems. One particular benefit is that sample application and testingmay be done in a relatively quick, easy, and simple manner. Further, asa result of the controlled flow provided by the present invention, lowvolume test samples may be accurately tested without the requirement ofcomplex and expensive equipment to obtain a useable sample. For example,whole blood drops having a volume of less than about 3 microliters maybe readily analyzed for the presence of an analyte in accordance withthe present invention.

While the invention has been described in detail with respect to thespecific embodiments thereof, it will be appreciated that those skilledin the art, upon attaining an understanding of the foregoing, mayreadily conceive of alterations to, variations of, and equivalents tothese embodiments. Accordingly, the scope of the present inventionshould be assessed as that of the appended claims and any equivalentsthereto.

1. A method for performing a lateral flow assay, the method comprising:providing a sampling membrane with a collection region and a detectionregion; defining a barrier between the collection region and thedetection region so that the collection region has a known saturationvolume that is less than about 100 microliters; saturating thecollection region with a test sample so that a known volume of the testsample is contained in the collection region that corresponds to theknown saturation volume of the collection region; removing the barrierbetween the collection region and detection region of the membrane; andsupplying a diluent to the collection region of the membrane tofacilitate flow of the test sample from the collection region to thedetection region of the membrane.
 2. The method of claim 1, wherein thebarrier is defined by a soluble composition in a defined band across thewidth of the membrane, the soluble composition present throughout themembrane in an amount to prevent migration of the test sample from thecollection region to the detection region of the membrane, and furthercomprising dissolving the soluble composition with the diluent.
 3. Themethod of claim 1, wherein the barrier is defined by a separation in themembrane, said step of removing the barrier comprising forming a bridgeacross the divide so that the test sample flows from the collectionregion to the detection region with the diluent.
 4. The method of claim3, wherein the test sample is whole blood, the bridging member alsofunctioning as a blood separation filter.
 5. The method of claim 1,further comprising supplying the test sample to a receiving region ofthe membrane, and further comprising defining the collection region byseparating the collection region from the receiving region prior tosupplying the diluent to the collection region.
 6. The method of claim5, wherein the collection region is separated from the receiving regionby scoring or cutting the membrane along a line that defines thecollection region.
 7. The method of claim 1, wherein the test sample iswhole blood, and further comprising separating plasma, serum, or bothfrom the whole blood for analysis.
 8. The method of claim 1, wherein thetest sample is conveyed to the collection region by defining a channelin the membrane.
 9. The method of claim 8, wherein a sampling tip isformed at an end of the channel, and further comprising placing the tipadjacent to the skin of a user for receipt of a test sample of blood.10. The method of claim 8, further comprising separating the channelfrom the collection region prior to supplying the diluent to thecollection region.
 11. The method of claim 8, wherein the channel isformed using one of a laser ablation or solvent microfabricationtechnique that renders at least a portion of the channel essentiallynon-conductive to the test sample.
 12. The method of claim 8, furthercomprising laminating the membrane to a support, and defining a bottomsurface of the channel with the support.
 13. The method of claim 8,further comprising treating the channel with a hydrophobic agent.